ADVANCES IN PLANT BREEDING & BIOTECHNOLOGY TECHNIQUES

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1 ADVANCES IN PLANT BREEDING & BIOTECHNOLOGY TECHNIQUES BOOK OF ABSTRACTS Pannonian Plant Biotechnology Association Conference for PhD Students in Plant Biology

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3 ADVANCES IN PLANT BREEDING & BIOTECHNOLOGY TECHNIQUES BOOK OF ABSTRACTS

4 Organized by: The Pannonian Plant Biotechnology Association in collaboration with Doctoral School of The Faculty of Agricultural and Food Sciences University of West Hungary, Mosonmagyaróvár Venue: The Faculty of Agricultural and Food Sciences University of West Hungary, Mosonmagyaróvár Date: April, National Development Agency The project is supported by the European Union and co-financed by the European Social Fund. TÁMOP /1/KONV Published by: Pannonian Plant Biotechnology Association Editor: Izabella Kőszegi Cover, layout: Csaba Maulis Printed by: Multiszolg Bt. Manager: Bálint Kajtor ISBN:

5 The aim of the conference for PhD students working in the field of plant biology is to exchange their ideas and their research work which is the basic for our future. PhD students are invited from all Doctoral Schools of the Universities for participation and presentations of their studies.

6 CONTENTS ORAL PRESENTATIONS DOMESTICATION BOTTLENECK AND GENETIC VARIETY OF GLUTENIN GENES IN WHEAT 10 S. Makai, L. Tamás, F. Békés, A. Juhász MOLECULAR BACKGROUND OF SPELT (T. SPELTA) PROTEINS AND THEIR ALLERGEN CHARACTERISTICS 12 A. Kovács, S. Tömösközi, F. Békés, L. Láng, A. Juhász, Z. Bedő PROTEOMIC ANALYSIS OF ALLERGENIC PROTEINS IN WHEAT 14 S. Fekecsová, M. Hajduch THE GENES OF AN ORGAN DEVELOPMENT REGULATING TRANSCRIPTION FACTOR FAMILY IN BRACHYPODIUM DISTACHYON 16 M. Gombos, Z. Zombori, M. Szécsényi, Gy. Sándor, J. Györgyey THE EFFECTS OF WHEAT GLUTENIN SUBUNITS ON THE FUNCTIONAL PROPERTIES OF RICE DOUGH 19 S. Pólya, G. Balázs, S. Tömösközi, F. Békés, L. Tamás, M. Oszvald GLOBALIZATION IN THE SEXUAL REPRODUCTION OF CEREALS 22 D. Polgári, L. Sági IDENTIFICATION OF MITOGEN-ACTIVATED PROTEIN KINASE SUBSTRATES IN ARABIDOPSIS PROTOPLAST TRANSIENT EXPRESSION SYSTEM 24 M. Dőry, Z. Doleschall, H. Ambrus, R. Dóczi REGULATION OF PROTECTIVE PROLINE SYNTHESIS IN ARABIDOPSIS THALIANA 26 D. Aleksza, H. Kovács, L. Szabados, G. V. Horváth FUNCTONAL ANALYSIS OF CANDIDATE VIRULENCE GENES OF PHYTOPATHOGEN FUNGUS VERTICILLIUM ALBO-ATRUM PREDICTED BY FUNGAL COMPARATIVE GENOMICS AND PROTEOMIC ANALYSIS 29 M. Flajšman, J. Jakše, S. Mandelc, S. Radišek, B. Javornik TRICHOMES TYPES AND THEIR DENSITY IN PARENTAL SPECIES SOLANUM TUBEROSUM AND S. CHACOENSE AND THEIR DERIVED SOMATIC HYBRIDS 32 A.-M. Mărgineanu, I. Molnár, E. Rakosy -Tican 4

7 THE EVALUATION OF RESISTANCE TO COLORADO POTATO BEETLE AND DETECTION OF GLYCOALKALOID CONTENT BY USING FOURIER TRANSFORM INFRARED SPECTROSCOPY IN SOLANUM TUBEROSUM + SOLANUM CHACOENSE SOMATIC HYBRIDS 34 I. Molnár, R. Boitor, R. Thieme, T. Thieme, E. Rakosy-Tican BIOPHYSICAL CHARACTERISATION AND IN VITRO DIGESTIBILITY OF SUNFLOWER STORAGE PROTEINS 36 B. Berecz, C. Mills, F. Láng, L. Tamás, P. Shewry, A. Mackie PROTEOMIC ANALYSIS OF FLAX ADAPTATION IN CHERNOBYL AREA USING GEL-FREE AND GEL-BASED APPROACH 38 D. Gábrišová, M. Danchenko, M.Hajduch RECENT ADVANCES IN STYRIAN OIL PUMPKIN BREEDING 40 K. Košmrlj, J. Murovec, D. Kastelec, A. Kladnik, B. Bohanec THE EVALUATION OF PROLINE, PLANT REGENERATION AND VIABILITY AFTER IN VITRO STRESS SELECTION SUSTAINS DROUGHT RESISTANCE IN POTATO MARKER-FREE TRANSGENIC LINES CARRYING PVYCP INTRON CONTAINING HAIRPIN CONSTRUCT 43 R. Mustata, I. Molnár, E. Rákosy-Tican TRANSGENIC PLANTS BENEFIT FROM REACTIVE ALDEHYDE DETOXIFYING AND SUGAR ALCOHOL PRODUCING FUNCTION OF THE SAME ALDO-KETO REDUCTASE ENZYME DURING HEAVY METAL, FREEZING AND SALT STRESS 45 Cs. Éva, H. Zelenyánszki, R. Tömösközi-Farkas, Á. Solti, L. Tamás EFFECT OF THE HARDENING UNDER DIFFERENT LIGHT CONDITIONS ON MAIZE CHILLING TOLERANCE 47 O. K. Gondor, M. Pál, T. Janda, G. Szalai THE EFFECTS OF DROUGHT ON PLANT DEFENCE SYSTEM IN WHEAT GENOTYPES WITH DIFFERENT SALICYLIC ACID CONTENT 49 V. Kovács, O. K. Gondor, I. Majláth, G. Szalai, T. Janda, M. Pál SOLID-STATE FERMENTATION AN ALTERNATIVE METHOD FOR ENRICHMENT OF AGRO-BASED MATERIALS WITH BIOACTIVE COMPOUNDS 51 L. Guothová, V. Matušovicová, T. Klempová, M. Čertík COLD RESPONSE AND GENE EXPRESSION PATTERN CHANGES IN TWO CHROMOSOME SUBSTITUTION LINES OF WHEAT (TRITICUM AESTIVUM L.) 54 B. Kalapos, G. Galiba, F. Marincs 5

8 UTILIZATION OF DIPLOID WHEATGRASSES FOR ALIEN CHROMOSOME DISCRIMINATION IN WHEAT AGROPYRON GLAEL HYBRID PROGENIES 56 K. Kruppa, É. Szakács, M. Molnár-Láng NOVEL UTILIZATION OF WILD RELATIVES IN BREAD WHEAT PREBREEDING 59 P. Mikó, M. Megyeri, M. Molnár-Láng DEVELOPMENT AND IDENTIFICATION OF NEW WHEAT-BARLEY DITELOSOMIC ADDITION LINES USING FLUORESCENCE IN SITU HYBRIDIZATION AND MOLECULAR MARKERS 62 E. Türkösi, A. Cseh, M. Molnár-Láng RESTRICTION FRAGMENT LENGTH POLYMORPHISM MAPPING OF FLAX GROWN IN RADIO-CONTAMINATED CHERNOBYL ENVIRONMENT SUGGESTS THE STABILITY OF DELTA-12 DESATURASE AND FATTY ACID DESATURASE 3C GENES 64 V. Lancíková, J. Žiarovská, M. Danchenko, V. Berezhna, M. Bežo, K. Ražná, N. M. Rashydov, M. Hajduch STRIGOLACTONES AND PARASITIC WEEDS 66 R. Matusova MOLECULAR DIVERSITY AND PHYLOGEOGRAPHY OF BEGOMOVIRUSES INFECTING VEGETABLE CROPS IN INDIA 68 B. S. Bhatt, A. K. Singh ANALYSIS OF NSS PROTEIN OF HUNGARIAN RESISTANCE-BREAKING TOMATO SPOTTED WILT VIRUS (TSWV) ISOLATES FROM PEPPER 70 Zs. Csömör, A. Almási, K. Nemes, K. Salánki, G. Csilléry, L. Palkovics, I. Tóbiás EFFECT OF MICROALGAE LEAF TREATMENTS ON SUNFLOWER GROWTH AND PRODUCTION 73 P. Pőthe, I. Gergely, V. Ördög EFFECT ON THE GROWTH, CONDITION, NUMBER OF QUALITY AND OTHER PARAMETERS OF PEPPER KALDOM BY USING NOSTOC ENTHOPHYTUM MICROALGAE-TREATMENT 76 J. Tóth, V. Ördög 6

9 POSTERS THE PLOIDY AND GENOME COMPOSITION OF SOLANUM TUBEROSUM AND SOLANUM BULBOCASTANUM SOMATIC HYBRIDS AND THEIR DERIVED BACKCROSS PROGENIES 80 T.-É. Dénes, I. Molnár, K. Kruppa, É. Szakács, M. Molnár-Láng, R. Thieme, E. Rákosy-Tican VARIAMETRIC ANALYSIS OF EINKORN (TRITICUM M. MONOCOCCUM) SEED POPULATIONS: MEASURING GENETIC DISTANCE USING PHENOVARIATION 82 A. Emődi, F. Gyulai, Z. Mravcsik, G. Gyulai, M. Megyeri, S. Vinogradov, T.A. Szabó, I. Rovner PRODUCING WHEAT/BARLEY INTROGRESSION LINES USING THE 2C GAMETOCID SYSTEM 85 D. Icsó, G. Linc, M. Molnár-Láng BIOTECHNOLOGICAL APPROACH TO CONTROL PARASITIC WEEDS 87 D. Kullačová, R. Matusova MOLECULAR CHARACTERIZATION OF NOSTOC AND ANABAENA MICROALGAE ISOLATES 89 N. Makra, G. Gell, A. Juhász, V. Soós, Z. Molnár, V. Ördög, E. Balázs GENOTYPE IDENTIFICATION OF ANCIENT VINEGRAPE (VITIS V. VINIFERA) SEED REMAINS (15 CENT. HUNGARY) BY DIGITAL MORPHOMETRY 91 Z. Mravcsik, F. Gyulai, A. Emődi, G. Gyulai, S. Vinogradov, I. Rovner LIGHT QUALITY INFLUENCES THE EXPRESSION LEVEL OF SOME CBF GENES 93 A. Novák, Á. Boldizsár, B. Tóth, G. Galiba SIGNIFICANTLY INCREASED GLUTATHIONE-S-TRANSFERASE ACTIVITY IN 35S (CAMV)-ZMGSTF4 TRANSGENIC ARABIDOPSIS THALIANA 95 K. Pilinszky, A. Bittsánszky, G. Gyulai, T. Kőmíves 7

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11 ORAL PRESENTATIONS

12 DOMESTICATION BOTTLENECK AND GENETIC VARIETY OF GLUTENIN GENES IN WHEAT S. Makai 1, 2, L. Tamás 1, F. Békés 3, A. Juhász 2 1 Doctocal School of Biology, Eötvös Loránd University, Budapest, Hungary 2 Agricultural Institute, MTA Centre for Agricultural Research, Martonvásár, Hungary 3 FBFD PTY LTD Beecroft, NSW, Australia Hexaploid wheat only exists in cultivated form and it is derived from a cross between the cultivated Triticum dicoccum and a wild goat grass. In one possible scenario its progenitors were as follows: Triticum urartu A genome, Aegilops speltoides B genome and Aegilops tauschii D genome. The progenitors of wheat acquired many morphological and physiological novelties, such as loss of seed shattering, increased yield, decreased chemical and morphological defenses, and uniformity in germination and growth phenology, erect growth to facilitate increased plant density in crop fields. All these are referred as domestication syndrome. Further traits were acquired during the cultivation process such as diversification in grain starch composition, and adaptation to different climates and latitudes and decrease in grain protein to carbohydrate ratio. Amongst the domesticated crops only hexaploid wheat went through speciation, all other plants retained their genetic relations to their wild types. Domesticated, hexaploid wheat has unique bread-making quality amongst the members of the Triticeae tribe. During the preparation of the bread dough, wheat flour is mixed with water that forms the gluten matrix, a giant, elastic and extensible polymer where the composing proteins are bound by intraand intermolecular disulfide bridges. The main components of wheat flour are high molecular weight glutenin, low molecular weight glutenin subunits (HMW GS and LMW GS), alpha-, gamma- and omega gliadins and starch. The ratio of prolamins to carbohydrates and the quality and composition of these prolamins in the seed directly influence the end-use quality; consequently all these properties have been greatly affected by the domestication event and the subsequent cultivation. One detrimental consequence of domestication is the decreased genetic diversity of genes related to domestication syndromes. This genetic restriction, also called genetic bottleneck, may restrict the possibilities of the breeder but can be overcome by backcrossing, as wild type plants present an unchartered genetic depositary. Indeed, haplotype analysis of Glu-1D genes reported less genetic diversity than their wild counterparts which is mainly due to the genetic bottleneck caused by human selection processes. Prolamins are specially expressed in the endosperm of the seed and are regulated via cis-regulatory elements. HMW GS proteins are encoded in the Glu-1 locus on the chromosome 1 of the three homoeolog genomes of hexaploid wheat. Due to an 10

13 ancient duplication event at Glu-1 locus, each locus contains two paralog glutenin genes, named as x and y type genes. Our earlier study reported that the promoters of Glu-1 genes have a conserved structure of 7 cis-regulatory modules (CRM) including the proximal basal promoter region. The motif composition of these modules varies across the x and y pairs as well as across the homeolog genes causing variation of activity between these genes. There are distinct differences between the modules of x and y type genes. We concluded that breeding at large did not influence the promoter architecture but rather the activity of the interacting transcription factors. In this study, we aim to analyze the role of domestication bottleneck that may have resulted the conserved, non-optimal (from the breeder s aspect) differences of promoter motif composition of the paralog Glu-1 genes. The analysis involved 156 promoter sequences of HMW GS from the Triticeae tribe. It contained 69 x-type and 76 y-type sequences. The modules of the promoter sequences were compared across species. Where information was available, the coding region were analyzed for their conserved features. The expression of x and y type HMW GS genes are different, x type genes always are expressed in higher amounts than their paralogs. In the case of A genome, y-type gene is generally silent in the hexaploid wheat. This can be explained either as a result of the breeding process or a simple heritage of the domestication events leading to hexaploid wheat. In case of the former, the stoichiometric ratio of x and y-type genes may represent an optimum for the end-use quality, thus it was the target of the selection process. This is supported by the fact that the numbers of cysteine and their distribution on the protein are characteristically different for x and y-type HMW GS. Since these cysteine residues play a crucial role in the development of the gluten matrix this aspect is not negligible, although needs a thorough examination. The other cause of the different expression level may lie in the fact that polyploidization is a relatively rare event. The diversity of HMW-GS genes were determined at this moment of speciation that represented a genetic bottleneck that narrowed the gene pool of the new specie. In this case, breeding could not broaden the diversity beyond a certain degree, thus we experienced low variability. This is supported by the fact that the diversity of wild type genes is higher than in the case of hexaploid wheat for both the cysteine distribution and promoter motif composition. However, there were more than one polyploidization event that lead to hexaploid wheat. First, the merge of the A and B genomes, then the merge of this tetraploid with the D genome. It seems very likely that the above described two possibilities both were at place but with different weight in these separate events. ACKNOWLEDGEMENT This work was supported by Hungarian Scientific Research Fund (OTKA K100881). 11

14 MOLECULAR BACKGROUND OF SPELT (T. SPELTA) PROTEINS AND THEIR ALLERGEN CHARACTERISTICS A. Kovács 1, S. Tömösközi 2, F. Békés 3, L. Láng 1, A. Juhász 1, Z. Bedő 1 1 Agricultural Institute, MTA Centre for Agricultural Research, Martonvásár, Hungary 2 Budapest University of Technology and Economics, Budapest, Hungary 3 FBFD PTY LTD Beecroft, NSW, Australia Wheat is one of those food components that are responsible for a large amount of food hypersensitivity reactions. There are a several type of wheat-related hypersensitivity (baker s asthma, IBS: irritable bowel syndrome, gluten intolarence). The most widespread diseases are wheat allergy and celiac disease triggered by certain proteins and their given short peptide sequences (epitopes) in wheat and a few other cereals (barley, rye, oat and triticale). The initial clinical symptoms of these disorders are often overlapping, that makes their differentiation and diagnosis difficult, however their pathogen mechanisms are absolutely different. The wheat allergy is an IgE-mediated reaction, pathological reaction of the immune system to wheat proteins.the celiac disease is an autoimmune disorders to prolamins (gluten proteins) of cereals. IgA/IgG are generated through the immune response. The prevalence of the celaic disease in the Caucasoid (Europid) race (inclusive of Hungary) is not more than 2%, until the rate of the population affected by any kind of cereal-related sensitivity/allergy with different severity and mechanism is more than 4%. In case of both disorders the information about the target molecules (antigens) are incomplete. The classical separation method of the wheat proteins is the Osborne solvent fractionation procedure. According to this alignment the watersoluble components are the albumins, the salt-soluble are the globulins, the prolamins, which are soluble in alcohol and the glutenins, which are only soluble in dilute acids or alkalis. The non-prolamin proteins (albumins and globulins) of wheat endosperm represent 20-25% of total grain proteins, the gliadins and glutenins constitute about 75-80%. Gluten is composed by interaction of the gliadins and glutenins. The main impact of the celiac disease is assigned to proteins member of the prolamin family. The epitopes responsible for allergenicity can be found both in the wheat prolamins and non-prolamin proteins. The quality and the composition of gluten proteins are very important from technological sight, determining the flour final utilization. So it is impossible to eliminate all of the toxic gluten protein fractions, because simultanously the quality of bread making would be degraded. In case of non gluten proteins, among sufficient number of examined genotypes particular non-allergic gene variants could be found. Previous research studies proved that, in spite of close genetic relationship among cultivars of wheat (T. aestivum) and spelt (T. spelta), spelt wheat resulted in better tolerance if products produced from its flour were consumed by people 12

15 suffering from wheat allergy. Differences mainly in water soluble proteins affected binding properties of IgE immunoglobulin in some European spelt cultivars. Similarily, products made from an Australian spelt genotype (GWF) possessing some mutations in a wheat allergy related expansin gene proved to be suitable to consume by patients. Based on these problems and possibilities the aim of our project was to develop new strategies for breeding selection of less or non-immune reactive wheat species. A range of experiments were set up, which aim was improving the appropriate identification and detection of IgE mediated immunogenic proteins and sequences in bread wheat and spelta genotypes. The protein composition of the examined European and Australian spelt cultivars was characterized by different separation methods such as chromatography: SE-HPLC, RP-HPLC, Lab-on-a-chip, and one dimensional SDS- and A-PAGE and two dimensional gelelectrophoresis. 4 different protein fraction (albumin, globulin, gliadin, glutenin) of the spelt varieties were characterized. The result of this protein profile analysis showed significant differences in both the prolamin and non prolamin profiles of wheat and spelt.the GWF Australian spelt genotype, which has less imune reactivity as indicated by previous clinical experiment, showed unique seed protein composition compared to other examined spelt genotypes. The SDS gel profile showed different ω-gliadin composition, some certain low molecular weight glutenins and γ-gliadins were absent. Another promising way for finding out the allergen potential of cereal varieties is the application of molecular marker based techniques. Molecular markers are identifiable DNA sequences, indicating the presence of certain allergenic/toxic proteins. PCR-based experiments were performed to characterise polymorphisms of T. aestivum and T. spelta genotypes and to highlight differences between species. Based on a publicly available research articles and databases IgE-binding sequences were selected (thioredoxin, expansin, α/β-gliadin, beta-purothionin, alpha-amylase inhibitor, different enzymes). A range of spelt and closely related species was screened using a set of identified allergen specific primers. Using these markers a multiplex PCR method was developed and used in marker assisted selection (MAS) in breeding programs in order to breed genotypes suitable for special needs. 13

16 PROTEOMIC ANALYSIS OF ALLERGENIC PROTEINS IN WHEAT S. Fekecsová 1, 2, M. Hajduch 1 1 Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Nitra, Slovakia 2 Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia Nowadays large study on plant proteins, proteomics, is one of the most dynamically developing field of scientific research. Proteomic as a discipline allows for comprehensive exploration of plant tissues and also providing a wide range of information about the molecule under investigation. Wheat (Triticum aestivum L.) is an important cereal grain for export and domestic consumption in many countries throughout the world. The main use of wheat grain is the production of flour which, depending on the specific type of wheat, is used in many baked goods (Tatham et al., 2008). Wheat proteins can be divided into two broad groups on the basis of their biological functions. Albumins and globulins are biologically active enzymes associated with nutritional quality of baked goods. Gluten proteins (gliadins and glutenins) are biologically inactive storage proteins making up about 80% of the total (Payne et al., 1985). The quality of bread and the other baked goods depends mainly on wheat s attributes like special storage proteins, gliadins and glutenins. However, these proteins are also connected with food and respiratory allergies (Baker s asthma) and intolerances (Coeliac disease) (Ciclitira et al., 2003) The aim of our work is to compare four genotypes of bread wheat (Triticum aestivum L.) based on the protein fractions using two-dimensional gel electrophoresis (2DE). This technique for quantitative proteome analysis combines protein separation by high-resolution (isoelectric focusing/sds-page) twodimensional gel electrophoresis (2DE) with mass spectrometry for identification of proteins spots. One of the most important method within two-dimensional gel electrophoresis is protein purification. The most important in terms of defining the precise protein is the correct extraction of the total mixture of proteins. Our goal is to optimize the best method for the extraction of protein fractions of wheat and get the best proteomic gels. Since each fraction cereal proteins are characterized by solubility in different solvents, it is necessary to create the best protocols for their perfect finish to a solution and then isolate the protein from the mixture. Albumins were extracted from 200 mg of a milled grain sample that was mixed continuously with 1ml of buffer containing 25 mm sodium phosphate (ph 7.5) for 60 min at 4 C. Gliadin proteins were extracted from 200 mg a milled grain sample using 1ml 50% (v/v) aqueous iso-propanol mixing at 1000 rpm for 30 min at room temperature. After mixing the both fraction of wheat were centrifuged at 5800 rpm for 15 min at room temperature. After centrifugation, supernatants were precipitation by 0.1M ammonium acetate in methanol and incubated at -20 C over night. On the second day pellet of proteins were washed twice with 0.1 M ammonium acetate 14

17 in methanol, twice with 80% (v/v) ice-cold acetone and finally with cold 70% (v/v) ethanol. Samples (50 µg protein from each fraction) were diluted in aliquot volume of IEF buffer (8 M urea, 2 M thiourea, 2% (w/v) CHAPS, 2% (v/v) Triton X-100, 50 mm dithiothreitol) and added 1,25 µl ampholyte and loaded into ph 5-8, 7 cm immobilized ph gradient strips (ReadyStrip IPG Strips BioRad) for the isoelectric focusing. For second dimension IPG strips were incubated in SDS equilibration buffer (1.5 M Tris-HCl ph 6.8, 6 M urea, 30% (v/v) glycerol, 5% (w/v) SDS for 15 min with 2% (w/v) dithiothreitol followed) followed by a second equilibration step of 15 min with the equilibration buffer containing 2.5% (w/v) iodoacetamide. The equilibrated strips were loaded on the top of 30% polyacrylamide gel. After the completion of 2DE gels were stained for 16 hour using Coomassie Brilliant Blue G-250 at room temperature. The presented experiments comparing different genotypes of wheat are only the part of big study about wheat proteins. The creation of two-dimensional protein gels of wheat grain will be followed by the analysis of individual protein spots excised and subjected to identification of the mass spectrometry. Data will be further processed by bioinformatics methods for the identification of proteins. The data collected will give us detailed information and comprehensive view of wheat protein. REFERENCES Ciclitira, P.J.- Moodie, S.J Coeliac disease. In Best Practice & Research Clinical Gastroenterology. ISSN , 2003, vol. 17, p Payne, P.I. Holt, L.M. Jarvis, M.G. Jackson, E.A Two-dimensional fractionation of the endospermproteins of bread wheat (Triticum aestivum): biochemical and genetic studies. In Cereal Chemistry. ISSN , 1985, vol. 62, p Tatham, A.S. Shewry, P.R Allergy to wheat and related cereals. In Clinical and Experimental Allergy. ISSN , 2008, vol. 38, p ACKNOWLEDGMENT This work was co-funded by European Community under project no : Building Research Centre AgroBioTech and project VEGA 2/0016/14 Proteomics mapping of clinically relevant proteins in wheat grain 15

18 THE GENES OF AN ORGAN DEVELOPMENT REGULATING TRANSCRIPTION FACTOR FAMILY IN BRACHYPODIUM DISTACHYON M. Gombos 1, Z. Zombori 2, M. Szécsényi 2, Gy. Sándor 2, J. Györgyey 2 1 PhD. School in Biology, University of Szeged, Hungary 2 Hungarian Academy of Sciences, Biological Research Centre, Institute of Plant Biology, Szeged, Hungary The impressive developmental capacity and morphological plasticity of plants are among the most important tokens of their adaptation to the continuously changing environmental conditions. During the accommodation process plants can modify their architecture in response to environmental stresses thanks to the dynamic regulation of meristematic cell activity. The molecular mechanisms regulating the boundaries between meristems and already differentiated organs are key components of this regulation process, and special cells at these boundaries take a crucial part in it. In accordance with their special function these cells express unique sets of genes, such as CUC (CUP SHAPED COTYLEDON), NAC (NO APICAL MERISTEMS) and LOB (LATERAL ORGAN BOUNDARIES DOMAIN) genes to form boundaries. The regulation of the meristematic activity through these genes is especially important for a proper and proportional organ development, including the formation of the whole root system (Rast and Rüdiger, 2008). Since the formation of efficient root architecture is essential for plants to survive under limited water supply, the main subject of our research was the better understanding the effects of the above mentioned molecular mechanisms on the root developmental processes via physiological and molecular biological tools. For this purpose we used purple false brome (Brachypodium distachyon) as a model in our experiments. Thanks to the simple growth requirements, short life cycle, fully sequenced and relatively small genome and owning to the close phylogenetic relationship to agronomical important cereals (eg. wheat, barley, rye), this annual grass has become an accepted and widely used model plant for monocots in the last 10 years (Vogel et al, 2010, Darper et al, 2001). Brachypodium is particularly useful in researches investigating monocot specific developmental processes, where Arabidopsis thaliana would not be appropriate model being a dicot plant. At first 31 various Brachypodium inbred lines were tested partially in rhizotrons and in pots in terms of their different responses to water limitation. On the basis of main component analysis it can be established that certain genotypes reply differently to drought stress in terms of general alterations in growth parameters and in root architecture. It was also revealed that deep-growing primary and nodal roots are essential for successful adaptation. 16

19 Among the candidates of root architecture influencing genes in this research we studied the LATERAL ORGAN BOUNDARIES-domain containing genes (LBDs) of Brachypodium. This gene family was first described in Arabidopsis thaliana as a plant specific transcription factor family. Their name refers to the observation that the spatial expression of the founding member of the family restricts typically to the boundaries between lateral organs (Shuai et al, 2002). In Arabidopsis thaliana 42 LOBdomain protein coding genes can be found. Their common feature is the presence of a conserved, 100 amino acid long LOB-domain structure which consist of a cysteine-rich CX 2 CX 6 CX 3 C motif suitable for DNA binding, a Gly-Ala-Ser box and a leucine-zipper like motif that allows protein-protein interactions (Matsumura et al, 2009). Although their exact functions are mainly unknown, on the basis of Arabidopsis experiments it has been demonstrated that LBD genes are involved in all aspects of plant development from embryogenesis to seed production owning to their crucial role in the lateral organ separation (Majer and Hochholdinger, 2011). In spite of the fact that their homologues can be found in almost all green organisms studied so far from more complex seaweeds to higher plants, aside from some exceptions we know hardly anything about their roles in monocots (Bortiri et al, 2006; Yoshiaki et al, 2005; Li et al, 2008). Therefore we aim to get to know in detail the processes controlled by LBD genes in Brachypodium distachyon, as a model system, with a major focus to root development. We could identify 24 LOB-domain protein coding genes in the genome of Brachypodium which can be divided into two major classes and some minor subclasses on the basis of their sequence homology. For the identification of root specific ones we analyzed the relative expression of LBD genes via quantitative realtime RT-PCR in 37 different organs and various parts of the plants from root tip as far as to shoot apex, both in vegetative and generative organs. According to the expression pattern characterization we found that certain LBD genes have distinct organ specificity: some of them are definitely active in flowers, in various parts of the floret and in developing seeds, while some others showed high activity in green plant parts, and of course there were some LBD genes which can be described as root specific ones. Generally speaking, aside from few exceptions the closely related LBD genes have similar expression pattern. In addition to that our analysis goes far beyond the similar literature dates in terms of its detailed resolution, we got several expression patterns which have a good correlation to the transcriptional activity of their homologues that can be found in other plants (eg. Arabidopsis, rice). This suggests an evolutionary conserved function of LBD genes. So far we selected two members from the 24 LBD genes for future analysis, namely the LBD15 and LBD13, for two reasons: at first, they could be associated presumably with cell cycle regulation on the basis of our unpublished experiments related to their homologues in Medicago truncatula. For second, in despite of their very close phylogenetic relationship they show a significantly different expression profile from each other (namely LBD13 showed a very sharp root tip specific expression, while the activity of LBD15 is less organ-specific, and its relative transcript level was especially high in generative organs). The promoter analysis 17

20 confirmed the root tip specificity of LBD13, apart from the fact that promoter-gfpreporter gene construction revealed activity also in the anthers; and the preliminary analysis of LBD15 overexpressing Brachypodium seedlings suggests that LBD15 may have effect on germination, spike development, shoot and root growth. ACKNOWLEDGMENT This research was supported by the Hungarian National Research Found (OTKA K76273, OTKA-K109719). 18

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